Yasuhiko Sekine

Evidence for in vivo ribosome recycling, the fourth step in protein biosynthesis

Ribosome recycling factor (RRF) catalyzes the fourth step of protein synthesis in vitro: disassembly of the post‐termination complex of ribosomes, mRNA and tRNA. We now report the first in vivo evidence of RRF function using 12 temperature‐sensitive Escherichia coli mutants which we isolated in this study. At non‐permissive temperatures, most of the ribosomes remain on mRNA, scan downstream from the termination codon, and re‐initiate translation at various sites in all frames without the presence of an initiation codon. Re‐initiation does not occur upstream from the termination codon nor beyond a downstream initiation signal. RRF inactivation was bacteriostatic in the growing phase and bactericidal during the transition between the stationary and growing phase, confirming the essential nature of the fourth step of protein synthesis in vivo.

Bacterial insertion sequences.

While DNA has a property of being fundamentally stable as invariable genetic information, studies on gene expression and gene organization have revealed that the genome is often subject to dynamic changes. Some of these changes are brought about by mobile genetic elements which have been found in prokaryotic and eukaryotic genomes so far studied. Insertion sequences (ISs) are bacterial mobile DNA elements which cause various kinds of genome rearrangements, such as deletions, inversions, duplications, and replicon fusions, by their ability to transpose. These were discovered during investigation of mutations that are highly polar in the galactose and lactose operons of Escherichia coli K−12 (Jordan etal. 1968; Malamy 1966, 1970; Shapiro 1969) and in the early genes of bacteriophage λ (Brachet et al. 1970). Many of these mutations were shown by electron microscope heteroduplex analysis to be insertions of distinct segments of DNA which are hence called insertion sequences (Fiandt et al. 1972; Hirsch et al. 1972; Malamy et al. 1972). It was later shown that the transcription of flanking genes can originate from promoters located within an IS or from hybrid promoters created by the insertion event or by the IS-mediated genome rearrangements. An important note here is that the finding of IS elements as mobile elements to new loci to turn genes either off or on would re-evaluate the controlling elements described in maize by McClintock (1956, 1965) (see Starlinger and Saedler 1976).

Role of ribosome recycling factor (RRF) in translational coupling

RNA phage GA coat and lysis protein expression are translationally coupled through an overlapping termination and initiation codon UAAUG. Essential for this coupling are the proximity of the termination codon of the upstream coat gene to the initiation codon of the lysis gene (either a <3 nucleotide separation or physical closeness through a possible hairpin structure) but not the Shine–Dalgarno sequence. This suggests that the ribosomes completing the coat gene translation are exclusively responsible for translation of the lysis gene. Inactivation of ribosome recycling factor (RRF), which normally releases ribosomes at the termination codon, did not influence the expression of the reporter gene fused to the lysis gene. This suggests the possibility that RRF may not release ribosomes from the junction UAAUG. However, RRF is essential for correct ribosomal recognition of the AUG codon as the initiation site for the lysis gene.

Involvement of H-NS in Transpositional Recombination Mediated by IS1

IS1, the smallest active transposable element in bacteria, encodes a transposase that promotes inter- and intramolecular transposition. Host-encoded factors, e.g., histone-like proteins HU and integration host factor (IHF), are involved in the transposition reactions of some bacterial transposable elements. Host factors involved in the IS1 transposition reaction, however, are not known. We show that a plasmid with an IS1 derivative that efficiently produces transposase did not generate miniplasmids, the products of intramolecular transposition, in mutants deficient in a nucleoid-associated DNA-binding protein, H-NS, but did generate them in mutants deficient in histone-like proteins HU, IHF, Fis, and StpA. Nor did IS1 transpose intermolecularly to the target plasmid in the H-NS-deficient mutant. The hns mutation did not affect transcription from the indigenous promoter of IS1 for the expression of the transposase gene. These findings show that transpositional recombination mediated by IS1 requires H-NS but does not require the HU, IHF, Fis, or StpA protein in vivo. Gel retardation assays of restriction fragments of IS1-carrying plasmid DNA showed that no sites were bound preferentially by H-NS within the IS1 sequence. The central domain of H-NS, which is involved in dimerization and/or oligomerization of the H-NS protein, was important for the intramolecular transposition of IS1, but the N- and C-terminal domains, which are involved in the repression of certain genes and DNA binding, respectively, were not. The SOS response induced by the IS1 transposase was absent in the H-NS-deficient mutant strain but was present in the wild-type strain. We discuss the possibility that H-NS promotes the formation of an active IS1 DNA-transposase complex in which the IS1 ends are cleaved to initiate transpositional recombination through interaction with IS1 transposase.

Inhibition of transpositional recombination by OrfA and OrfB proteins encoded by insertion sequence IS3

An insertion element IS3 is flanked by terminal inverted repeat (IR) sequences. IS3 encodes two, out‐of‐phase, overlapping open reading frames, orfA and orfB, from which three proteins are produced. OrfAB is a transframe protein produced by −1 translational frameshifting between orfA and orfB, and it is known to be IS3 transposase. OrfA and OrfB are the proteins produced without frameshifting, but their functions have not been elucidated.

Identification of the site of translational frameshifting required for production of the transposase encoded by insertion sequence IS 1.

SummaryPrevious genetic analyses indicated that translational frameshifting in the −1 direction occurs within the run of six adenines in the sequence 5′-TTAAAAAACTC-3′ at nucleotide positions 305–315 in IS 1, where the two out-of-phase reading frames insA and B′-insB overlap, to produce transposase with a polypeptide segment Leu-Lys-Lys-Leu at residues 84–87. IS 1 mutants with a 1 by insertion, which encode mutant transposases with an amino acid substitution within the polypeptide segment at residues 84–87, did not efficiently mediate cointegration, except for an IS 1 mutant which encodes a mutant transposase with a Leu-Arg-Lys-Leu segment instead of Leu-LysLys-Leu. An IS 1 mutant with the DNA segment 5′-CTTAAAAACTC-3′ at positions 305–315 carrying the termination codon TAA in the B′-insB reading frame could still mediate cointegration, indicating that codon AAA for Lys corresponding to second, third and fourth positions in the run of adenines is the site of frameshifting. The β-galactosidase activity specified by several IS 1- lacZ fusion plasmids, in which B′-insB is in-frame with lacZ, showed that the region 292–377 is sufficient for frameshifting. The protein produced by frameshifting from the IS 1-lacZ plasmid in fact contained the polypeptide segment Leu - Lys - Lys - Leu encoded by the DNA segment 5′-TTAAAAAACTC-3′, indicating that −1 frameshifting does occur within the run of adenines.

Presence of a Characteristic D-D-E Motif in IS1 Transposase

Transposases encoded by various transposable DNA elements and retroviral integrases belong to a family of proteins with three conserved acidic amino acids, D, D, and E, constituting the D-D-E motif that represents the active center of the proteins. IS1, one of the smallest transposable elements in bacteria, encodes a transposase which has been thought not to belong to the family of proteins with the D-D-E motif. In this study, we found several IS1 family elements that were widely distributed not only in eubacteria but also in archaebacteria. The alignment of the transposase amino acid sequences from these IS1 family elements showed that out of 14 acidic amino acids present in IS1 transposase, three (D, D, and E) were conserved in corresponding positions in the transposases encoded by all the elements. Comparison of the IS1 transposase with other proteins with the D-D-E motif revealed that the polypeptide segments surrounding each of the three acidic amino acids were similar. Furthermore, the deduced secondary structures of the transposases encoded by IS1 family elements were similar to one another and to those of proteins with the D-D-E motif. These results strongly suggest that IS1 transposase has the D-D-E motif and thus belongs to the family of proteins with the D-D-E motif. In fact, mutant IS1 transposases with an amino acid substitution for each of the three acidic amino acids possibly constituting the D-D-E motif were not able to promote transposition of IS1, supporting this hypothesis. The D-D-E motif identified in IS1 transposase differs from those in the other proteins in that the polypeptide segment between the second D and third E in IS1 transposase is the shortest, 24 amino acids in length. Because of this difference, the presence of the D-D-E motif in IS1 transposase has not been discovered for some time.

DNA sequences required for translational frameshifting in production of the transposase encoded by IS 1

SummaryThe transposase encoded by insertion sequence IS 1 is produced from two out-of-phase reading frames (insA and B′-insB) by translational frameshifting, which occurs within a run of six adenines in the −1 direction. To determine the sequence essential for frameshifting, substitution mutations were introduced within the region containing the run of adenines and were examined for their effects on frameshifting. Substitutions at each of three (2nd, 3rd and 4th) adenine residues in the run, which are recognized by tRNALys reading insA, caused serious defects in frameshifting, showing that the three adenine residues are essential for frameshifting. The effects of substitution mutations introduced in the region flanking the run of adenines and in the secondary structures located downstream were, however, small, indicating that such a region and structures are not essential for frameshifting. Deletion of a region containing the termination codon of insA caused a decrease in β-galactosidase activity specified by the lacZ fusion plasmid in frame with B′-insB. Exchange of the wild-type termination codon of insA for a different one or introduction of an additional termination codon in the region upstream of the native termination codon caused an increase in β-galactosidase activity, indicating that the termination codon in insA affects the efficiency of frameshifting.

Transposition of IS1 circles

IS1, the smallest active transposable element in bacteria, encodes transposase. IS1 transposase promotes transposition as well as production of miniplasmids from a plasmid carrying IS1 by deletion of the region adjacent to IS1. The IS1 transposase also promotes production of IS1 circles consisting of the entire IS1 sequence and a sequence, 6–9u2003bp in length, as a spacer between terminal inverted repeats of IS1. The biological significance of the generation of IS1 circles is not known.

Isolation and characterization of IS1 circles.

Transposase encoded by insertion sequence IS1 is produced from two out-of-phase reading frames by translational frameshifting that occurs in a run of adenines. An IS1 mutant with a single adenine insertion in the run of adenines efficiently produces transposase, resulting in generation of miniplasmids by deletion for a region adjacent to IS1 from a plasmid carrying the IS1 mutant. Here, we found that besides miniplasmids, cells harboring the plasmid contained minicircles without the region required for replication. Cloning and DNA sequencing of the minicircles revealed that most of them were IS1 circles consisting of the entire IS1 sequence and a sequence, 5-9 bp in length, which intervenes between terminal inverted repeats, IRL and IRR, of IS1. Analysis of more IS1 circles isolated by polymerase chain reaction revealed that the intervening sequence was derived from the region flanking either IRL or IRR in the parental plasmid, suggesting that IS1 circles are generated by an excision event from the parental plasmid. The IS1 circles may be formed due to the cointegration reaction occurring within the parental plasmid carrying IS1.